Our lab is constantly searching for the elusive combination of Southern hybridization protocols that will result in rapid turnover, good signals, and repetitive use of membranes. We would like to describe our latest attempt to reach hybridization nirvana, adapting the "modified dry blot" protocol of Kempter and co-workers (TIG 7:109-110, 1991) for RFLP analysis.
Digested genomic DNA (approximately 10µg of DNA/40µl/lane) is electrophoresed through a 0.8% agarose gel, in 1 X RBE running buffer (40mM Tris-Acetate, pH 7.3, 2mM Na2 EDTA, 0.02mM NaOAc, 32mM glacial acetic acid). After electrophoresis, the gel is stained in ethidium bromide and photographed. The gel is prepared for DNA transfer using 2 X gel volumes of solution for each step: 1) depurinate in 0.25N HCl for 20 min; 2) denature twice in 0.4M NaOH, 15 min each; 3) neutralize once in 0.25M Tris-Acetate, 0.1M NaCl, pH 8.0 for 15 min; and 4) incubate in the 0.025M Tris-Acetate, 0.1M NaCl, pH 8.0 transfer solution for 15 min. Slowly agitate gel during treatment using a rotary platform, and rinse the gel in ddH2O between each treatment (except after transfer solution).
Cut 5 pieces of Whatman 3MM chromatography paper and one piece of Gene Screen Plus hybridization membrane (DuPont) the same size as the gel. Soak one piece of 3MM paper in the transfer solution and lay on plastic wrap. Lay the gel onto the 3MM paper (doesn't matter which side of the gel is up) and gently roll out air bubbles using a pipet. Pre-wet the hybridization membrane in ddH2O and equilibrate for 15 min in transfer solution. Place the membrane on top of the gel, soak a second piece of 3MM paper in transfer solution and stack on top of the membrane. Gently roll out air bubbles with a pipet and place the four remaining dry pieces of 3MM paper on top of the stack. Cut a 2" (2.5cm) stack of paper towels slightly larger than the gel. Place towels on top and add 1kg of weight (one Sigma Chemical Co. catalog does nicely) on top of the stack. The minimum time for transfer is 1 hr, but we routinely go 2 hrs or overnight depending upon the time of day. After 2 hrs transfer, DNA is not detected in the gel. The membrane can be dried either at room temperature or in vacuo at 80 C for 2 hrs.
The membrane is pre-hybridized (5 X SSC, 2 X Denhardt's, 50mM Tris-HCl, pH 8.0, 5mM Na2 EDTA, 0.5% sarcosine, 1.0ml of 10mg/ml boiled salmon sperm) in a 65 C water bath for a minimum of 3 hrs. We use Dazey Micro-Seal pouches (8" X 12", 2MILS thick), sealing the pouches with a ClampCo sealer (Fisher Sci.). We normally use 30 to 40ml of solution per pouch, with 1 to 8 membrane(s)/pouch. The pre-hybridization solution is replaced with hybridization solution (5 X SSC, 2 X Denhardt's, 50mM Tris-HCl, pH 8.0, 5mM Na2 EDTA, 0.5% sarcosine, 10% dextran sulphate, 1.0ml of 10mg/ml boiled salmon sperm). A G-50 purified random-primed 32P-dCTP probe (100ng isolated insert or linearized plasmid, 20µCi 32P-dCTP; Feinberg and Vogelstein, Anal. Biochem. 132:6-13, 1983) is boiled for 5 min and added to the hybridization pouch. Air bubbles are removed and the sealed pouch is incubated 24 to 48 hr in a 65 C water bath. The membranes are washed five times for 5 min each in 2 X SSC, 0.1% SDS at 65 C (400ml/wash), then an additional five times in 0.2 X SSC, 0.1% SDS at 65 C. The membranes are wrapped in Saran wrap and autoradiography is performed as described in Maniatis et al. (Molecular Cloning, CSH Laboratory Press, 1982) for 24 to 48 hrs. Membranes are stripped in 0.4M NaOH for 30 min and neutralized in 0.4M Tris-HCl, 0.1 X SSC, and 0.1% SDS for 30 min. The membranes are returned to pre-hybridization solution or wrapped in Saran Wrap to prevent drying.
Starting from DNA digestion, we have reduced the amount of time to detect single-copy maize sequences with 32P-dCTP nick translocated probes to a minimum of four days. The Gene Screen Plus membrane was found to be superior to Zeta-Probe and Hybond-N, measured by signal intensity and background remaining after stripping the membranes. We have stripped and re-hybridized Gene Screen Plus membranes eight or more times maintaining signal intensity without increasing background. The hybridization solution is re-used to probe additional membranes. After use, the hybridization solution is stored in a 50ml Falcon tube at -20 C. The used hybridization solution is added directly to the pouch, along with 1.0ml of 10mg/ml boiled salmon sperm, and incubated for 2 days at 65 C. The resulting hybridization signal is comparable to the original hybridization up to 2 weeks after initial hybridization. This technique was developed with considerable input from Mark Jones and Brenda Schult (USDA, ARS, Wooster, OH 44691).
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